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STAT1 modification improves therapeutic effects of interferons on lung cancer cells.

Chen J, Zhao J, Chen L, Dong N, Ying Z, Cai Z, Ji D, Zhang Y, Dong L, Li Y, Jiang L, Holtzman MJ, Chen C - J Transl Med (2015)

Bottom Line: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness.STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China. jiestory414@163.com.

ABSTRACT

Background: Interferons (IFNs) have potent anti-proliferative, pro-apoptotic, and immunomodulatory activities against cancer. However, the clinical utility of IFNs is limited by toxicity and pharmacokinetics making it difficult to achieve sustained therapeutic levels especially in solid tumors.

Methods: Signal Transducer and Activator of Transcription 1 (STAT1) or a modified STAT1 (designated STAT1-CC) that is hyper-responsive to IFN were overexpressed in lung cancer SPC-A-1 and H1299 cells using lentiviral vectors. Transduction efficiency was monitored using enhanced green fluorescent protein (EGFP) expression. After transduction, cells were treated with interferon-gamma (IFN-γ) or interferon-beta (IFN-β) and monitored for cell proliferation, migration, and invasiveness using Cell Counting Kit-8 and transwell chamber assays and for apoptosis using Annexin V detection by flow cytometry. In addition, levels of STAT1, STAT1 Tyr-701 phosphorylation (pSTAT1), fibronectin, and β-catenin were determined using western blotting. In the case of IFN-γ stimulation, levels of S100A4, proliferating cell nuclear antigen (PCNA), and c-fos expression were also determined.

Results: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness. Moreover, STAT1 and STAT1-CC expression caused increases in pSTAT1 and decreases in fibronectin and β-catenin levels. STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.

Conclusion: The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells. Results from this study suggest that combined treatment of IFNs and STAT1-CC might be a feasible approach for the clinical management of lung cancer in the future.

No MeSH data available.


Related in: MedlinePlus

Overexpression of STAT1 or STAT1-CC induces growth inhibition of lung cancer cells. a Western blotting revealed whole cell levels of STAT1 and pSTAT1 in SPC-A-1 and H1299 cells transduced with lenti-STAT1 or lenti-STAT1-CC. b Overexpression of STAT1 or STAT1-CC resulted in significant reductions in the proliferation of SPC-A-1 and H1299 cells (++P < 0.01, lenti-STAT1 vs lenti-EGFP; **P < 0.01, lenti-STAT1-CC vs lenti-EGFP). c, d Representative image of foci formation in a monolayer culture. The colony formation rates were significantly decreased in STAT1 and STAT1-CC-expressed SPC-A-1 and H1299 cells. Experiments were repeated at least three times. Data are shown as mean ± SEM. **P < 0.01
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Fig2: Overexpression of STAT1 or STAT1-CC induces growth inhibition of lung cancer cells. a Western blotting revealed whole cell levels of STAT1 and pSTAT1 in SPC-A-1 and H1299 cells transduced with lenti-STAT1 or lenti-STAT1-CC. b Overexpression of STAT1 or STAT1-CC resulted in significant reductions in the proliferation of SPC-A-1 and H1299 cells (++P < 0.01, lenti-STAT1 vs lenti-EGFP; **P < 0.01, lenti-STAT1-CC vs lenti-EGFP). c, d Representative image of foci formation in a monolayer culture. The colony formation rates were significantly decreased in STAT1 and STAT1-CC-expressed SPC-A-1 and H1299 cells. Experiments were repeated at least three times. Data are shown as mean ± SEM. **P < 0.01

Mentions: In order to overexpress wild type STAT1 and STAT1-CC mutant in lung cancer cells, we inserted these cDNA containing IRES-EGFP sequences into a lentiviral vector, and at the same time set up EGFP alone as a control using the same vector (Fig. 1). The transfection efficiencies (EGFP positive cells) of the stable EGFP-, STAT1- or STAT1-CC-expressing SPC-A-1 and H1299 cells were more than 95 % by flow cytometry analysis. Expression level of STAT1 or STAT1-CC was verified by western blot analysis. Interestingly, higher levels of pSTAT1 were detected in STAT1-CC transduced SPC-A-1 and H1299 cells at baseline compared to STAT1 transduced cells (Fig. 2a). Cell viability was determined using Cell Counting Kit-8 (CCK-8). As shown in Fig. 2b, STAT1 or STAT1-CC inhibited SPC-A-1 and H1299 lung cancer cell growth under basal culture conditions. Moreover, colony formation was significantly decreased in SPC-A-1 and H1299 cells transduced with STAT1 or STAT1-CC when compared to those in EGFP-expression cells. Colony formation rate for SPC-A-1 cells was 62.9 % in the EGFP controls and in cells transduced with STAT1 or STAT1-CC decreased to 24.7 and 22.6 % respectively. In H1299 cells, colony formation rate was reduced from 82.9 % in EGFP controls to 42.5 % with STAT1 and 40.1 % with STAT1CC cells (Fig. 2c, d). These data indicated that overexpression of STAT1 or STAT1-CC in lung cancer SPC-A-1 and H1299 cells inhibited cell proliferation and colony formation.Fig. 2


STAT1 modification improves therapeutic effects of interferons on lung cancer cells.

Chen J, Zhao J, Chen L, Dong N, Ying Z, Cai Z, Ji D, Zhang Y, Dong L, Li Y, Jiang L, Holtzman MJ, Chen C - J Transl Med (2015)

Overexpression of STAT1 or STAT1-CC induces growth inhibition of lung cancer cells. a Western blotting revealed whole cell levels of STAT1 and pSTAT1 in SPC-A-1 and H1299 cells transduced with lenti-STAT1 or lenti-STAT1-CC. b Overexpression of STAT1 or STAT1-CC resulted in significant reductions in the proliferation of SPC-A-1 and H1299 cells (++P < 0.01, lenti-STAT1 vs lenti-EGFP; **P < 0.01, lenti-STAT1-CC vs lenti-EGFP). c, d Representative image of foci formation in a monolayer culture. The colony formation rates were significantly decreased in STAT1 and STAT1-CC-expressed SPC-A-1 and H1299 cells. Experiments were repeated at least three times. Data are shown as mean ± SEM. **P < 0.01
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Fig2: Overexpression of STAT1 or STAT1-CC induces growth inhibition of lung cancer cells. a Western blotting revealed whole cell levels of STAT1 and pSTAT1 in SPC-A-1 and H1299 cells transduced with lenti-STAT1 or lenti-STAT1-CC. b Overexpression of STAT1 or STAT1-CC resulted in significant reductions in the proliferation of SPC-A-1 and H1299 cells (++P < 0.01, lenti-STAT1 vs lenti-EGFP; **P < 0.01, lenti-STAT1-CC vs lenti-EGFP). c, d Representative image of foci formation in a monolayer culture. The colony formation rates were significantly decreased in STAT1 and STAT1-CC-expressed SPC-A-1 and H1299 cells. Experiments were repeated at least three times. Data are shown as mean ± SEM. **P < 0.01
Mentions: In order to overexpress wild type STAT1 and STAT1-CC mutant in lung cancer cells, we inserted these cDNA containing IRES-EGFP sequences into a lentiviral vector, and at the same time set up EGFP alone as a control using the same vector (Fig. 1). The transfection efficiencies (EGFP positive cells) of the stable EGFP-, STAT1- or STAT1-CC-expressing SPC-A-1 and H1299 cells were more than 95 % by flow cytometry analysis. Expression level of STAT1 or STAT1-CC was verified by western blot analysis. Interestingly, higher levels of pSTAT1 were detected in STAT1-CC transduced SPC-A-1 and H1299 cells at baseline compared to STAT1 transduced cells (Fig. 2a). Cell viability was determined using Cell Counting Kit-8 (CCK-8). As shown in Fig. 2b, STAT1 or STAT1-CC inhibited SPC-A-1 and H1299 lung cancer cell growth under basal culture conditions. Moreover, colony formation was significantly decreased in SPC-A-1 and H1299 cells transduced with STAT1 or STAT1-CC when compared to those in EGFP-expression cells. Colony formation rate for SPC-A-1 cells was 62.9 % in the EGFP controls and in cells transduced with STAT1 or STAT1-CC decreased to 24.7 and 22.6 % respectively. In H1299 cells, colony formation rate was reduced from 82.9 % in EGFP controls to 42.5 % with STAT1 and 40.1 % with STAT1CC cells (Fig. 2c, d). These data indicated that overexpression of STAT1 or STAT1-CC in lung cancer SPC-A-1 and H1299 cells inhibited cell proliferation and colony formation.Fig. 2

Bottom Line: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness.STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, Zhejiang, China. jiestory414@163.com.

ABSTRACT

Background: Interferons (IFNs) have potent anti-proliferative, pro-apoptotic, and immunomodulatory activities against cancer. However, the clinical utility of IFNs is limited by toxicity and pharmacokinetics making it difficult to achieve sustained therapeutic levels especially in solid tumors.

Methods: Signal Transducer and Activator of Transcription 1 (STAT1) or a modified STAT1 (designated STAT1-CC) that is hyper-responsive to IFN were overexpressed in lung cancer SPC-A-1 and H1299 cells using lentiviral vectors. Transduction efficiency was monitored using enhanced green fluorescent protein (EGFP) expression. After transduction, cells were treated with interferon-gamma (IFN-γ) or interferon-beta (IFN-β) and monitored for cell proliferation, migration, and invasiveness using Cell Counting Kit-8 and transwell chamber assays and for apoptosis using Annexin V detection by flow cytometry. In addition, levels of STAT1, STAT1 Tyr-701 phosphorylation (pSTAT1), fibronectin, and β-catenin were determined using western blotting. In the case of IFN-γ stimulation, levels of S100A4, proliferating cell nuclear antigen (PCNA), and c-fos expression were also determined.

Results: We found that expression of STAT1 or STAT1-CC enhanced the effect of IFN-γ and, IFN-β on inhibition of human lung cancer cell proliferation, migration and invasiveness. Moreover, STAT1 and STAT1-CC expression caused increases in pSTAT1 and decreases in fibronectin and β-catenin levels. STAT1-CC showed increased effects compared to STAT1 on IFN-γ induced pSTAT1 and down-regulation of S100A4, PCNA, and c-fos levels.

Conclusion: The results show that STAT1-CC exhibited more strength in improving the antitumor response of IFNs in lung cancer cells. Results from this study suggest that combined treatment of IFNs and STAT1-CC might be a feasible approach for the clinical management of lung cancer in the future.

No MeSH data available.


Related in: MedlinePlus